The present invention is directed to methods of purification of Hepatitis A Virus from the supernatant of an infected cell culture and production of a preparation of purified HAV antigen. The present invention is also directed to an HAV vaccine composition comprising a preparation consisting of purified mature HAV particles in an amount sufficient to induce a protective immune response in a mammal.
Hepatitis A continues to cause sporadic cases, endemics and occasional deaths and is a public health problem all over the world. The infection is caused by Hepatitis A Virus (HAV) a member of the picornavirus family, a group of small non-enveloped RNA viruses. The virus particle is 27-32 nm in diameter and is composed of three polypeptides VP1, VP2 and VP3, cleaved from a single polypeptide precursor molecule.
Hepatitis A Virus (HAV) is the only hepatotropic virus which can be isolated from cell culture, but the virus is usually difficult to propagate, with long incubation period and no cytopathic effect. Even tough several primate cell types have been reported to support replication of HAV, such as fetal rhesus monkey kidney cell line (FRhk-4), primary African green monkey kidney cells (AGKM), continuous African green monkey kidney cells (BCS-1), these cannot generally be used for human vaccine because monkey kidneys often have high content of latent simian viruses which may become apparent in the course of virus production for vaccine. Other cell lines cannot be used because of the tumorogenic nature of some of these cells invokes constraints against their use for vaccine production. Mass production of primary human epithel, fibroblast or kidney cells or cell strains to propagate HAV is limited by the low passage number of these cells in culture. In fact, the applicable guidelines of the World Health organization (WHO) indicate that only a few cell lines are allowed for virus vaccine production.
One of the cell lines which is currently accepted and validated for the production of a vaccine applicable to humans are VERO cells. VERO cells are continuous monkey kidney cells that have been licensed for use in the manufacturing process of human vaccines and are currently used for the production of poliomyelitis and rabies vaccine. Attempts have also been made to use VERO cells for HAV production, but replication of HAV on VERO cells is limited because VERO cells have a temperature restriction of viral growth and virus is never found in the supernatant fluids of infected cells (Locarnini et al., 1981, J. Virol. 37: 216-225). U.S. Pat. No. 4,783,407 discloses the production of HAV on VERO cells in roller bottles at a temperature no higher than 33xc2x0 C. to overcome the temperature restriction. In this system about 50 xcexcg HAV antigen can be obtained per roller bottle followed by freeze-thawing of the cultured cells. A commercial vaccine based on propagation of HAV on VERO cells has never been described.
So far, formalin inactivated HAV vaccines have been produced for clinical trials (Andre et al., 1990, In: Melnick (ed): Prog. Med. Virol. Basel, Karger 37: 72-95, Armstrong et al, 1993, J. Hepatology 18:20-26) and four have been licensed, which induce long-lasting immunity and protection from primary infection. The manufacturing process of the currently available inactivated HAV whole virus vaccines uses the human embryonic lung fibroblast cell line MRC-5 as host cells which grow slowly in tissue culture and only by addition of fetal calf serum.
The problems arising from the use of serum in the cell culture and/or protein additives derived from an animal or human source added to the culture medium, i.e. the varying quality and composition of different batches and the risk of contamination with mycoplasma, viruses or BSE-agent, are well-known. Therefore, many attempts are being made to provide efficient host systems and cultivation conditions that do not require serum or other serum derived compounds. Besides, avoidance of contamination by the use of serum free medium would allow more efficient purification due to less contamination in the starting material comprising the viral antigen.
Binn et al. (1984. J. Clincal. Microbiol. 20: 28-33) tested several primate cell types for replication of HAV and optimal conditions for isolation and production of quantities of virus. Serum free production of HAV on BSC-1 cells, a heterodiploid cell line that until now has not been used for preparation of vaccines for use in humans, in roller flasks revealed that after 21 days of the cultivation process virus antigen can be found in the supernatant and the cell fraction. Cells maintained in serum free medium supported viral growth equal to those maintained in serum. A candidate HAV vaccine obtained by low speed centrifugation of supernatant of freeze-thawed cells and supernatant fluid of infected BSC-1 cells maintained in serum free medium was described by Binn et al., 1986 (J. Infect. Diseases 153: 749-756).
Flehmig et al. (1987. J. Medical Virol. 22:7-16) prepared candidate HAV vaccine with HAV isolated from cell culture supernatant of persistently infected normal human embryonic fibroblasts grown in serum containing medium which had shown no cytopathic effect. Thereby, HAV antigen isolated and purified in serial steps from large amounts of supernatants produced in NUNC cell factories was used for vaccination tests.
However, all strains of HAV which have been grown in cell culture are characterized by inefficient release of virus into the culture supernatant. Although as much as 50% of infectious virus may be released, typically less than 30% of infectious virus is extracellular (Nasser et al., 1987. Appl. Environmental Microbiol. 53: 2967-2971). Therefore, antigen is often undetectable in unconcentrated culture supernatant and the concentrations of large volumes contributes to difficulties in HAV purification with this process. Because HAV antigen is not efficiently released into the culture supernatant and methods to concentrate the large volume are costly (Bishop et al., 1994. J. Virol. Meth. 47:203-216), most purification processes described uses HAV antigen from cell lysate of intracellularly produced virus as source for production of HAV vaccine (EP 0 339 667; EP 0 583 142, Andre et al., 1990, In: Melnick (ed): Prog. Med. Virol. Basel, Karger 37: 72-95; Armstrong et al, 1993, J. Hepatology 18:20-26); Hagen et al., 1996, Biotechnol. Appl. Biochem. 23:209-215; Bader et al., 1996, Amer. J. Gastroenterol. 91:217-222, Hennessey et al., 1999, Vaccine 17:2830-2885, WO 00/23574). However, these processes are time-consuming, make use of detergent necessary to release intracellularly produced antigen from the cells and need intense and serial purification steps to remove detergent and contaminants derived from the cells.
For the induction of a protective immune response it is suggested that the capsid proteins must be folded and assembled in the right confirmation and that the precursor proteins are not capable to elicit a protective immune response. Mature HAV particles consist of three virus capsid proteins (VP1, VP2, VP3). These proteins are derived from a single precursor molecule (P1) by several sequential cleavages. During virus maturation and assembly different intermediates are formed. The pre-cleaved proteins assemble first to a pentameric structure and then 60 pentamers form a provirion. The provirions consist of VP1, VP0 and VP3. The mature virus particles derive from these provirions after a last maturation cleavage of VP0 in VP2 and VP4. VP4 is not present in the mature virus particle.
HAV large scale preparations from the cell lysates and/or the cell culture supernatants contain mixed populations of mature virions, provirions and procapsids (Bishop et al., 1997. Arch. Virol. 142:2147-2160; EP 339 667, EP 339 668). The mature virus is composed of polypeptides VP1, VP2 and VP3, wherein the capsid proteins VP1 and VP3 contain the major antigenic sites and are capable to induce neutralizing antibodies (Lemon et al., 1989, In: Semler et al. eds. Molecular aspects of picornavirus and detection. Washington, D.C.: ASM p 193-208). Attempts have been made to purify HAV and separate the different forms of HAV particles. Bishop et al. 1997 (supra) used linear gradient centrifugation to separate the different HAV particle forms and found that HAV particles at a density of 1.32-1.33 g/cm3 are a mixture of VP0- and VP2-containing particles indicating an incomplete separation of provirions and mature virions. In the fractions containing predominantly mature HAV virions more VP2 than VPO in a ration of 55% to 45%, respectively, are found. Both virions and provirions are detected in cell lysates and culture supernatant and, in addition to VP0, released particles containing variable levels of VP1 precursor protein PX, having a molecular weight of about 67 KD, was detected. Dubois et al. (1991. J. Virol. Meth. 32:327-334) prepared a vaccine from a major peak fraction at a density of 1.33 g/cm3 comprising complete particles purified by isopycnic centrifugation. U.S. Pat. No. 5,268,292 described isolation and purification of HAV from persistently infected cells, and found that most of silver stained protein is accounted for by the viral polypeptides VP1, VP2 and VP3, but also a polypeptide of about 67 kD was detectable.
The worldwide market demand for HAV vaccines is on the order of 100 Million doses per year. Efficient vaccine production requires the growth of large-scale quantities of virus produced in high yields from a host system. Moreover, there is a need for an approach to viral propagation, employing materials that are already available and requiring a minimal number of time-consuming manipulations, wherein the selection of a combination of host cells, culture medium, growth conditions and production system is essential to achieve an efficient production process. Most vaccines have not been purified to preserve sensitive biological activity which is critical to the efficacy of the vaccine. A pure product would be expected to produce more a consistent vaccine having higher immunogenicity and produce fewer side effects from a clinical point of view.
It is an object of the present invention to provide a method of purification of HAV from cell culture supernatant of an infected cell culture.
It is another object of the invention to provide for a method of isolation of complete HAV particles from the supernatant of an infected cell culture.
It is another object of the invention to provide for a method of isolation of mature HAV particles from the supernatant of an infected cell culture.
It is another object of the present invention to provide a method for production of purified preparation of HAV particles.
It is also an object of the invention to provide a method for production of a purified preparation consisting of complete HAV particles
It is also an object of the invention to provide for a method for production of a purified preparation consisting of mature HAV particles.
It is an object of the present invention to provide a method for production of preparation consisting of inactivated, purified mature HAV particles.
It is also an object of the invention to provide a preparation consisting of purified HAV particles.
It is another object of the invention to provide a vaccine purified complete HAV antigen.
It is another object of the invention to provide a vaccine purified mature HAV antigen.